CN103724424B - mPEG-SPA-pGLP-2 compound as well as preparation method and application thereof - Google Patents
mPEG-SPA-pGLP-2 compound as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an mPEG-SPA-pGLP-2 compound as well as a preparation method and application thereof. The compound is formed by connecting porcine glucagon-like peptide-2 (pGLP-2) and methoxy polyethylene glycol-succinimidyl propionic acid ester (mPEG-SPA) through an addition reaction, wherein the Lys on the 30th site of the pGLP-2 and end double bonds of the mPEG-SPA form a covalent bond to be connected. The compound is a single-point modification product separated from a modified mixture through weak-acid cation-exchange resin chromatography. The mPEG-SPA-pGLP-2 compound is single in modification product and simple in separation and purification.
Description
Technical field
The invention belongs to the modification mixture of peptide or protein and the technical field of preparation thereof, be specifically related to pig glucagon-like-peptide-2 (the porcine glucagon-like peptide-2 that a kind of mono methoxy polyethylene glycol is modified, pGLP-2), i.e. a kind of mono methoxy polyethylene glycol-pig glucagon-like-peptide-2 mixture and its preparation method and application.
Background technology
Glucagon-like-peptide-2 (glucagon-like peptide-2, GLP-2) is one of 33 amino acid whose hyperglycemic-glycogenolytic factor derived peptide of Proglucagon genetic transcription, translation aftertreatment processing, primarily of the L emiocytosis of distal ileum and colon.GLP-2 is promoted enteric epithelium propagation, suppression enteric epithelium apoptosis by specificity, is increased many-sided structure recovery and barrier function improvement promoting damage intestinal mucosa such as the confession of enteron aisle blood, gastric acid secretion inhibiting, reduction intestinal permeability, and effect is better than other the non-specific intestines somatomedins found in the past, for the damage for the treatment of intestine of young pigs and dysfunction provide wide Research Prospects.PGLP-2 and hGLP-2 has similar enteron aisle growth promoting function, and homology is 82%, is extended, containing 35 amino acid by C end.
But in pig body, dipeptidyl peptidase-IV (dipeptidyl peptidase-IV, the DPP-IV) fast degradation extensively existed in the easy body of pGLP-2 falls the first two amino acid His of N end
1-Ala
2, Half-life in vivo is 8.4min, seriously constrains it and applies.
Polyoxyethylene glycol (PEG) is a kind of hydrophilic, uncharged linear macromolecule; protein is after PEG modifies, and molecular weight increases, and the filtration of renal glomerulus reduces; the barrier action of PEG protects protein not easily by protease hydrolysis, contributes to the prolongation of protein drug transformation period.But the separation and purification of albumen PEG modified outcome is all more difficult.Reason is as follows: (1) albumen is after PEG modifies, and many physico-chemical properties there occurs change, as iso-electric point, molecular mass, solubleness, settling ratio etc.; (2) PEG molecule has the conformation of stretching, extension in aqueous, and its hydrodynamic volume is far longer than the globular preteins of same molecular mass, makes very difficult with being separated of PEG; (3), there is certain molecular vibrational temperature in the unhomogeneity of PEG molecule, even if therefore comprise the same protein of identical PEG number, its relative molecular weight is also incomplete same; (4) when the modifiable amino acid sites of protein surface is more, PEG molecule can be combined on multiple different amino-acid residue, makes the space structure of PEG modified protein complicated further.More than for albumen PEG modifies and the core of product development and difficult point place.
At present, modify by PEG the research that pGLP-2 extends its transformation period treatment intestine of young pigs disease and have no report.
Summary of the invention
For the advantage only containing a Lys residue in pGLP-2, first object of the present invention is to provide a kind of mPEG-SPA-pGLP-2 mixture, and this mixture obtains single-point modified outcome, thus avoids the polymorphism of decorating site, makes subsequent purification easy.MPEG-SPA-pGLP-2 mixture remains the biologic activity of pGLP-2, and compared with pGLP-2, Increased Plasma Half-life, can be applied to the treatment of intestinal tract disease more easily.Second object of the present invention is to provide the preparation method of above-mentioned a kind of mPEG-SPA-pGLP-2 mixture.3rd object of the present invention is to provide the pharmaceutical applications of above-mentioned a kind of mPEG-SPA-pGLP-2 mixture.
In order to realize first above-mentioned object, present invention employs following technical scheme:
A kind of mPEG-SPA-pGLP-2 mixture, by pig glucagon-like-peptide-2 (porcine glucagon-like peptide-2, pGLP-2) with mono methoxy polyethylene glycol-succinimide propionic acid acid esters (methoxy-polyethylene glycol-succinimidyl-propionic acid ester, mPEG-SPA) connected and composed by addition reaction, the Lys that described pGLP-2 is the 30th forms covalent linkage with the terminal double link of described mPEG-SPA and is connected.
As preferably, the aminoacid sequence of described pGLP-2 is: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH
2.
As preferably, the molecular weight of described mono methoxy polyethylene glycol-succinimide propionic acid acid esters is 5 ~ 20KD.
In order to realize second above-mentioned object, present invention employs following technical scheme:
Prepare a method for the mPEG-SPA-pGLP-2 mixture described in above-mentioned arbitrary technical scheme, the method comprises the following steps:
1) pGLP-2 is prepared into the solution that pH is 7.5 ~ 9.0;
2) pGLP-2 solution step 1) prepared and mPEG-SPA react, and the molar concentration rate of described pGLP-2 and mPEG-SPA is 1:1 ~ 1:8, and temperature of reaction is 4 ~ 37 DEG C, and the reaction times is 0.5 ~ 24 hour;
3) separation and purification, obtains mPEG-SPA-pGLP-2 mixture.
As preferably, pGLP-2 is dissolved in the Tris-HCl damping fluid of 50 mmol/L pH 7.5 ~ 9.0 by described step 1), and final concentration is 1.2 mg/mL.
As preferably, described step 2) in add 1% TFA termination reaction.
As preferably, described step 3) separation and purification adopts weakly acidic cation-exchange resin chromatography; Preferred again, described weakly acidic cation-exchange resin adopts C M Sepharose Fast Flow chromatography column.
As preferably, by step 2) crude product solution prepared dilutes 10 times with the acetate buffer solution of 20 mmol/L pH 4.0, upper C M Sepharose Fast Flow chromatography column, the gradient elution of 0% to 100% is carried out with the acetate buffer solution of the 20 mmol/L pH 4.0 containing 1 mol/L NaCl, flow velocity is 1 mL/min, is the elute soln of the 2nd and the 3rd absorption peak in 215 nm place collection, 3 absorption peaks at wavelength; The Millipore Amicon Ultra super filter tube by the elute soln molecular weight cut-off collected being 3 ~ 10KD is concentrated, and removes NaCl, lyophilize.
When common polyethylene glycol modified protein reacts, owing to containing multiple Methionin with free amine group in protein, multiple spot modified outcome can be formed in polyoxyethylene glycol reaction, such as, containing 2 Methionins in GLP-1, the free amine group that can provide has 2, can form the mix products of two modified outcomes and mono-modified product, bring a difficult problem to subsequent purification and quality control.PGLP-2 of the present invention only has a Lys, and by the optimization of reaction conditions, obtain single-point modified outcome, separation and purification is simple.MPEG-SPA-pGLP-2 mixture remains the biologic activity of pGLP-2, compared with pGLP-2, Increased Plasma Half-life, a shot can mouse colitis that significantly DSS causes sexually revise, make to be administered once and become possibility, the treatment of intestinal tract disease can be applied to more easily.
Accompanying drawing explanation
Fig. 1 is the C M Sepharose F F chromatographic separation purifying figure of mPEG-SPA-pGLP-2 mixture.Wherein, 1 is penetrate peak, and 2 is the absorption peak of mPEG-SPA, and 3 is absorption peaks of single-point modified outcome mPEG-SPA-pGLP-2.
Fig. 2 mPEG-SPA-pGLP-2 mixture and pGLP-2 are to the vitro enzyme Numerical solution (n=5) of DPP-IV enzyme.PGLP-2 (◆) and mPEG-SPA-pGLP-2 (■) and DPP-IV react at 37 ° of C, detect, with peak area content during t=0 as 100% at the time point RP-HPLC preset.
Fig. 3 is the HE colored graph of mouse Colon inflammatory change.
Fig. 4 is the inflammatory scoring of mouse Colon (n=6).The non-parametric Wilcoxon rank test of data acquisition, do not have same letter represent significant difference (
p< 0.05).
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.MPEG-SPA used, chemical reagent etc. in below implementing, and the experimental technique of unreceipted actual conditions, undertaken routinely or by the condition that goods supplier is advised.
the preparation of embodiment 1 mPEG-SPA-pGLP-2 mixture
In the present embodiment, the structural formula of pGLP-2 mixture is: pGLP-2(1-35), sequence is as follows: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH
2.The molecular weight of mPEG-SPA is 5KD.
The first step, is dissolved in 1.2mg pGLP-2 in the Tris-HCl damping fluid of 1mL 50 mmol/L pH 7.5 ~ 9.0.
Second step, in the solution of the first step gained, add 6.0mg, molecular weight is the mPEG-SPA of 5KD, mixes.
3rd step, carries out mPEG modification reaction 0.5 ~ 24h at the solution of second step gained is placed in 4 ~ 30 DEG C.
4th step, adds the TFA termination reaction of 1% by the solution through the 3rd step process, obtain mPEG-SPA-pGLP-2 mixture crude product solution.
5th step, get the crude product solution that the 4th step obtains and dilute 10 times with the acetate buffer solution of 20 mmol/L pH 4.0, upper C M Sepharose Fast Flow chromatography column, the gradient elution of 0% to 100% is carried out with the acetate buffer solution of the 20 mmol/L pH 4.0 containing 1 mol/L NaCl, flow velocity is 1 mL/min, is the elute soln of the 2nd and the 3rd absorption peak in 215 nm place collection, 3 absorption peaks at wavelength.
Illustrate, see in Fig. 1, figure, 1 is first absorption peak in 3 absorption peaks, namely penetrates peak, and 2 and 3 is the 2nd and the 3rd absorption peak in 3 absorption peaks respectively, and 2 is mPEG-SPA, and 3 is single-point modified outcome mPEG-SPA-pGLP-2.
6th step, is that the Millipore Amicon Ultra super filter tube of 3KD concentrates by the elute soln molecular weight cut-off that the 5th step is collected, and removes NaCl, lyophilize.The molecular weight obtaining 0.4mg is the mPEG-SPA-pGLP-2 mixture sterling of 8873Da.
Illustrate, the mPEG-SPA-pGLP-2 mixture sterling distilled water that the 6th step is obtained redissolves, and is prepared into the solution of 1mg/mL.Getting 1 μ L uses UltrafleXtreme MALDI-TOF-MS instrument to carry out molecular weight determination, and result is shown in Fig. 1, and in Fig. 1, peak 3 is mPEG-SPA-pGLP-2 that single-point is modified.
the vitro enzyme Numerical solution of embodiment 2 mPEG-SPA-pGLP-2 mixture
The first step, is dissolved in 10 mmol/L by mPEG-SPA-pGLP-2 or pGLP-2 of 25 nmol/L, the triethylamine-HCI buffer of pH 7.4.
Second step, adds the DPP-IV enzyme (Sigma-Aldrich) of 100 mU/mL, hatches for 37 DEG C in the solution of the first step, and the time point 10% TFA termination reaction preset, RP-HPLC detects the residual content of mPEG-SPA-pGLP-2 or pGLP-2.
Explanation, RP-HPLC testing conditions is: use Agilent company 1200 of U.S. liquid chromatograph, ZORBAX SB-C18 pillar (4.6 mm × 250 mm, 5 μm), automatic sampler sample size is 20 μ L, flow velocity 1 mL/min, mobile phase A is the deionized water containing 0.1% TFA, Mobile phase B is the acetonitrile containing 0.1% TFA, Mobile phase B 38% to 50% wash-out 15 min, 215 nm monitorings.
Illustrate, result is see in Fig. 2, figure, and transformation period of mPEG-SPA-pGLP-2 is the transformation period of 296.3min, pGLP-2 is 18.3min, illustrates that mPEG-SPA-pGLP-2 mixture can extend the transformation period of pGLP-2.
the intestinal regeneration effect of embodiment 3 mPEG-SPA-pGLP-2 mixture
Experiment material and method:
Male and healthy BALB/C mice (cleaning grade, Zhejiang Academy of Medical Sciences animal experimental center provides);
Paraffin slicing machine (German Leica company), fluorescent microscope pick up camera (Japanese OLYMPUS company BX20 type), automatic dehydrator (Thermo shandon company of Britain);
Male and healthy BALB/C mice, is divided into 4 groups.1st group, DSS(molecular weight 36000 – 50000, purchased from Shanghai MP company) control group; 2nd group, DSS+pGLP-2 group; 3rd group, DSS+mPEG-SPA-pGLP-2 group; 4th group, drinking-water group, all mixture mPEG-SPA-pGLP-2 are the object product prepared in embodiment 1.In the present embodiment, the dosage of each group is in table 1.
The dosage that table 1 is respectively organized
1st ~ 3 groups of mouse drink 3% DSS 1st ~ 9 day every day, within the 10th day, drink distilled water, and the 4th group of mouse freely drinks distilled water in 1st ~ 10 days, the NaCl solution group of the 10th day the 1st, 4 group of abdominal injection 300 μ L 0.9%; The NaCl solution of the 2nd group of abdominal injection 300 μ L 0.9% and 30 μ g pGLP-2; The NaCl solution of the 3rd group of abdominal injection 300 μ L 0.9% and 30 μ g mPEG-SPA-pGLP-2.11st day, weigh after 12h on an empty stomach, cervical dislocation puts to death mouse, opens abdominal cavity along ventrimeson, take out intestinal tissue, measuring from pyloric region to returning the total small intestinal length of cecum and caecum end to the colon lengths of anus, getting colon, putting into 10% neutral formalin liquid, carry out routine paraffin wax section, after HE dyeing, under microscope 50 times of visuals field, selection is determined the intestinal tissue region of complete fine hair and takes pictures, and carries out the scoring of colon inflammatory.
Result as shown in Figure 3, the HE colored graph of DSS group mouse Colon, its pathology is the heaviest, the obvious oedema of colon intestines wall thickens, and mucous layer mucomembranous epithelial cell destroys, and goblet cell obviously reduces, crypts extensive damage, form ulcer surface, proper mucous membrane, submucosa is the interior a large amount of inflammatory cell infiltration of muscle layer even.The HE colored graph of DSS+pGLP-2 group mouse Colon, its pathology is taken second place, and mucous layer mucomembranous epithelial cell destroys, and goblet cell obviously reduces, and crypts destroys serious, proper mucous membrane, and submucosa is the interior a large amount of inflammatory cell infiltration of muscle layer even.DSS+mPEG-SPA-pGLP-2 group mouse Colon intestines wall Mild edema, mucomembranous epithelial cell is complete, and goblet cell reduces, and crypts destroys on a small quantity, a small amount of inflammatory cell infiltration in lamina propria.The HE colored graph of drinking-water group mouse Colon, for normal colonic tissue's structure is clear, have no and thicken, mucomembranous epithelial cell is complete, and in lamina propria, inflammatory cell is accidental, and goblet cell has no minimizing and crypts has no destruction.As shown in Fig. 4 histogram numerical value be 6 mouse colon inflammatory scoring mean value, compared with drinking-water group, DSS process significantly increase colon inflammatory mark (
p< 0.001), and inject mPEG-SPA-pGLP-2 group can significantly (
p< 0.001) generation of inflammation-inhibiting, substantially reach Normal Colon level.The inflammatory lesion that injection pGLP-2 causes DSS also makes a significant impact (
p< 0.001), but still there were significant differences to suppress the effect of inflammatory and colon normal configuration.Illustrate that the mouse Colon inflammatory disease that shot mPEG-SPA-pGLP-2 can effectively suppress DSS to cause becomes, action effect is obviously better than injecting pGLP-2.
Protection content of the present invention is not limited to above embodiment.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.
Claims (11)
1. a mPEG-SPA-pGLP-2 mixture, it is characterized in that: connected and composed by addition reaction by pig glucagon-like-peptide-2 (pGLP-2) and mono methoxy polyethylene glycol-succinimide propionic acid acid esters (mPEG-SPA), the Lys that described pGLP-2 is the 30th forms covalent linkage with the terminal double link of described mPEG-SPA and is connected.
2. a kind of mPEG-SPA-pGLP-2 mixture according to claim 1, is characterized in that: the aminoacid sequence of described pGLP-2 is: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH
2.
3. a kind of mPEG-SPA-pGLP-2 mixture according to claim 1 and 2, is characterized in that: the molecular weight of mono methoxy polyethylene glycol-succinimide propionic acid acid esters is 5 ~ 20KD.
4. prepare a method for the mPEG-SPA-pGLP-2 mixture described in claim 1 ~ 3 any one claim, it is characterized in that the method comprises the following steps:
1) pGLP-2 is prepared into the solution that pH is 7.5-9.0;
2) pGLP-2 solution step 1) prepared and mPEG-SPA react, and the mol ratio of described pGLP-2 and mPEG-SPA is 1:1 ~ 1:8, and temperature of reaction is 4 ~ 37 DEG C, and the reaction times is 0.5 ~ 24 hour;
3) separation and purification, obtains mPEG-SPA-pGLP-2 mixture.
5. the preparation method of mPEG-SPA-pGLP-2 mixture according to claim 4, is characterized in that: pGLP-2 is dissolved in the Tris-HCl damping fluid of 50 mmol/L pH 7.5 ~ 9.0 by step 1), and final concentration is 1.2 mg/mL.
6. the preparation method of mPEG-SPA-pGLP-2 mixture according to claim 4, is characterized in that: step 2) in add 1% TFA termination reaction.
7. the preparation method of mPEG-SPA-pGLP-2 mixture according to claim 4, is characterized in that: step 3) separation and purification adopts weakly acidic cation-exchange resin chromatography.
8. the preparation method of mPEG-SPA-pGLP-2 mixture according to claim 7, is characterized in that: described weakly acidic cation-exchange resin adopts C M Sepharose Fast Flow chromatography column.
9. the preparation method of mPEG-SPA-pGLP-2 mixture according to claim 7, it is characterized in that: by step 2) crude product solution prepared dilutes 10 times with the acetate buffer solution of 20 mmol/L pH 4.0, upper C M Sepharose Fast Flow chromatography column, the gradient elution of 0% to 100% is carried out with the acetate buffer solution of the 20 mmol/L pH 4.0 containing 1 mol/L NaCl, flow velocity is 1 mL/min, is the elute soln of the 2nd and the 3rd absorption peak in 215 nm place collection, 3 absorption peaks at wavelength; The Millipore Amicon Ultra super filter tube by the elute soln molecular weight cut-off collected being 3 ~ 10KD is concentrated, and removes NaCl, lyophilize, and the 3rd absorption peak is mPEG-SPA-pGLP-2 mixture.
10. the mPEG-SPA-pGLP-2 mixture in claim 1-3 described in any one is for the preparation of the application in the disorderly medicine for the treatment of intestines function of piglings.
MPEG-SPA-pGLP-2 mixture in 11. claim 1-3 described in any one is for the preparation of the application in treatment chitling tract disease medicine.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676163A (en) * | 2004-09-30 | 2005-10-05 | 华东师范大学 | Human glucagon-like peptide-1 compound and its preparing method |
| CN101709085A (en) * | 2009-11-02 | 2010-05-19 | 山东泰邦生物制品有限公司 | Polyethylene glycol modified human serum albumin and preparation method thereof |
| WO2011088837A1 (en) * | 2010-01-20 | 2011-07-28 | Zealand Pharma A/S | Treatment of cardiac conditions |
| WO2012155780A1 (en) * | 2011-05-19 | 2012-11-22 | 江苏豪森药业股份有限公司 | Branched-peg modified glp-1 analogue and pharmaceutically acceptable salts thereof |
| CN103333238A (en) * | 2013-06-18 | 2013-10-02 | 华东师范大学 | mPEG-MAL-aGLP-1 composite |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676163A (en) * | 2004-09-30 | 2005-10-05 | 华东师范大学 | Human glucagon-like peptide-1 compound and its preparing method |
| CN101709085A (en) * | 2009-11-02 | 2010-05-19 | 山东泰邦生物制品有限公司 | Polyethylene glycol modified human serum albumin and preparation method thereof |
| WO2011088837A1 (en) * | 2010-01-20 | 2011-07-28 | Zealand Pharma A/S | Treatment of cardiac conditions |
| WO2012155780A1 (en) * | 2011-05-19 | 2012-11-22 | 江苏豪森药业股份有限公司 | Branched-peg modified glp-1 analogue and pharmaceutically acceptable salts thereof |
| CN103333238A (en) * | 2013-06-18 | 2013-10-02 | 华东师范大学 | mPEG-MAL-aGLP-1 composite |
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